Detection of miRNA serum markers for liver cirrhosis and its application
A serum and application technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the problem that the role of microRNA cannot be underestimated
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[0073] The preparation of the miRNA chip can adopt conventional manufacturing methods of biochips known in the art. For example, if the solid phase carrier is a modified glass slide or silicon wafer, and the 5' end of the probe contains amino-modified poly dT strings, the oligonucleotide probe can be formulated into a solution, and then spotted with a spotting instrument. The miRNA chip of the present invention can be obtained by arranging in a predetermined sequence or array on a modified glass slide or a silicon chip, and then fixing it by standing overnight. If the nucleic acid does not contain amino modification, its preparation method can also refer to: "Gene Diagnosis Technology-Non-radioactive Operation Manual" edited by Wang Shenwu; J.L.erisi, V.R.Iyer, P.O.BROWN. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science, 1997; 278: 680 and Ma Liren, edited by Jiang Zhonghua. Biochip. Beijing: Chemical Industry Press, 2000, 1-130.
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Embodiment 1
[0093] sample source
[0094] Sera were collected from 30 cases of liver cirrhosis and 15 cases of normal people from the Department of Gastroenterology, Xinhua Hospital. All the above-mentioned specimens were obtained with the approval of the ethics committee of the WHO partner organization authorized by the Shanghai Municipal Government. The clinical data of tissue samples include: gender, age, pathological grade, etc.
Embodiment 2
[0096] Serum marker screening
[0097] miRNA extraction: use Ambion's mirVana TM Isolation kit extraction was performed according to the kit instructions of the company, and the specific operation was as follows: take 600 μl of serum, add 500 μl of miRNA lysate, mix by inverting, and place on ice for 10 minutes. Add an equal volume of acid-phenol-chloroform mixture, vortex mix, centrifuge at 10,000 rpm for 5 min at room temperature, and take the upper aqueous phase. Add one-third volume of absolute ethanol to the above organic extract. Mix well and add to collection column. Centrifuge at 10,000rpm for 15s. Collect the effluent liquid, add two-thirds of the volume of absolute ethanol, and mix well. The mixture was added to a new column, centrifuged at 10,000 rpm for 15 s, and the effluent was discarded. Add 700 μl wash solution 1 to the column and centrifuge for 15s. Add 500μl wash solution 2 / 3 to the column respectively, centrifuge for 15s, and repeat once. Place the c...
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